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(ref. 3) and MYCN4 but found no mutations in any of these Mutations in the cyclin family (Supplementary Methods online). Next, we carried out genome- member FAM58A cause wide high-resolution oligonucleotide array comparative genomic hybridization (CGH)5 analysis (Supplementary Methods)ofgenomic an X-linked dominant disorder DNA from the most severely affected individual (case 1, with lower lid coloboma, epilepsy and syringomyelia) and identified a hetero- characterized by syndactyly, zygous deletion of 37.9–50.7 kb on Xq28, which removed exons 1 and 2ofFAM58A (Fig. 1i,j). Using real-time PCR, we confirmed the telecanthus and anogenital deletion in the child and excluded it in her unaffected parents (Supplementary Fig. 1a online, Supplementary Methods and and renal malformations Supplementary Table 1 online). Through CGH with a customized Sheila Unger1,2,12, Detlef Bo¨hm3,12, Frank J Kaiser4, oligonucleotide array enriched in probes for Xq28, followed by break- Silke Kaulfu5, Wiktor Borozdin3, Karin Buiting6, point cloning, we defined the exact deletion size as 40,068 bp 5 1 1 (g.152,514,164_152,554,231del( X, NCBI Build 36.2);

http://www.nature.com/naturegenetics Peter Burfeind , Johann Bo¨hm , Francisco Barrionuevo , Alexander Craig1, Kristi Borowski7, Kim Keppler-Noreuil7, Fig. 1j and Supplementary Figs. 2,3 online). The deletion removes Thomas Schmitt-Mechelke8, Bernhard Steiner9, Deborah Bartholdi9, the coding regions of exons 1 and 2 as well as intron 1 (2,774 bp), Johannes Lemke9, Geert Mortier10, Richard Sandford11, 492 bp of intron 2, and 36,608 bp of 5¢ sequence, including the 5¢ UTR Bernhard Zabel1,2, Andrea Superti-Furga2 &Ju¨rgen Kohlhase3 and the entire KRT18P48 pseudogene (NCBI ID 340598). Paternity was proven using routine methods. We did not find deletions We identified four girls with a consistent constellation of overlapping FAM58A in the available copy number variation facial dysmorphism and malformations previously reported in (CNV) databases. a single mother–daughter pair. Toe syndactyly, telecanthus and Subsequently, we carried out qPCR analysis of the three anogenital and renal malformations were present in all affected other affected individuals (cases 2, 3 and 4) and the mother-daughter individuals; thus, we propose the name ‘STAR syndrome’ for pair from the literature (cases 5 and 6). In case 3, we detected this disorder. Using array CGH, qPCR and sequence analysis, a de novo heterozygous deletion of 1.1–10.3 kb overlapping exon 5 we found causative mutations in FAM58A on Xq28 in all (Supplementary Fig. 1b online). Using Xq28-targeted array CGH

Nature Publishing Group Group 200 8 Nature Publishing affected individuals, suggesting an X-linked dominant and breakpoint cloning, we identified a deletion of 4,249 bp © inheritance pattern for this recognizable syndrome. (g.152,504,123_152,508,371del(chromosome X, NCBI Build 36.2); Fig. 1j and Supplementary Figs. 2,3), which removed 1,265 bp We identified four unrelated girls with anogenital and renal malfor- of intron 4, all of exon 5, including the 3¢ UTR, and 2,454 bp mations, dysmorphic facial features, normal intellect and syndactyly of of 3¢ sequence. toes. A similar combination of features had been reported previously We found heterozygous FAM58A point mutations in the remaining in a mother–daughter pair1 (Table 1 and Supplementary Note cases (Fig. 1j, Supplementary Fig. 2, Supplementary Methods online). These authors noted clinical overlap with Townes-Brocks and Supplementary Table 1). In case 2, we identified the mutation syndrome but suggested that the phenotype represented a separate 555+1G4A, affecting the splice donor site of intron 4. In case autosomal dominant entity (MIM601446). Here we define the 4, we identified the frameshift mutation 201dupT, which immediately cardinal features of this syndrome as a characteristic facial appearance results in a premature stop codon N68XfsX1. In cases 5 and 6, with apparent telecanthus and broad tripartite nasal tip, variable we detected the mutation 556-1G4A, which alters the splice syndactyly of toes 2–5, hypoplastic labia, anal atresia and urogenital acceptor site of intron 4. We validated the point mutations malformations (Fig. 1a–h). We also observed a variety of other and deletions by independent rounds of PCR and sequencing features (Table 1). or by qPCR. We confirmed paternity and de novo status of On the basis of the phenotypic overlap with Townes-Brocks, the point mutations and deletions in all sporadic cases. None Okihiro and Feingold syndromes, we analyzed SALL1 (ref. 2), SALL4 of the mutations were seen in the DNA of 60 unaffected female

1Institute of Human Genetics, 2Centre for Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg, D-79106 Freiburg, Germany. 3Center for Human Genetics Freiburg, D-79100 Freiburg, Freiburg, Germany. 4Institut fu¨ r Humangenetik, Universita¨tsklinikum Schleswig-Holstein, Campus Lu¨ beck, D-23538 Lu¨ beck, Germany. 5Institut fu¨ r Humangenetik, Universita¨tGo¨ttingen, D-37073 Go¨ttingen, Germany. 6Institut fu¨ r Humangenetik, Universita¨tsklinikum Essen, D-45122 Essen, Germany. 7Division of Medical Genetics, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242, USA. 8Abteilung Neuropa¨diatrie, Kinderspital, CH-6000 Luzern, Switzerland. 9Institut fu¨ r Medizinische Genetik, Universita¨tZu¨ rich, CH-8603 Schwerzenbach, Switzerland. 10Center for Medical Genetics, Ghent University Hospital, B-9000 Ghent, Belgium. 11Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 OXY, UK. 12These authors contributed equally to this work. Correspondence should be addressed to J.K. ([email protected]). Received 10 October 2007; accepted 2 January 2008; published online 24 February 2008; doi:10.1038/ng.86

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controls, and no larger deletions involving FAM58A were found in 93 unrelated array-CGH investigations. By analyzing X-chromosome inactivation (Supplementary Meth- syndrome ods and Supplementary Fig. 4 online), we found complete skewing Townes-Brocks of X inactivation in cases 1 and 3–6 and almost complete skewing in case 2, suggesting that cells carrying the mutation on the active

XX have a growth disadvantage during fetal development. Okihiro

syndrome Using RT-PCR on RNA from lymphoblastoid cells of case 2 (Supple- Occasional X mentary Fig. 2), we did not find any aberrant splice products as additional evidence that the mutated allele is inactivated. Further- more, FAM58A is subjected to X inactivation6. In cases 1 and 3, the Feingold syndrome parental origin of the deletions could not be determined, as a result of 1 individual lack of informative SNPs. Case 5, the mother of case 6, gave birth to two boys, both clinically unaffected (samples not available). We cannot exclude that the condition is lethal in males. No fetal losses were

3rd reported from any of the families. o

VU reflux The function of FAM58A is unknown. The gene consists of five coding exons, and the 642-bp coding region encodes a of 214 amino acids. GenBank lists a mRNA length of 1,257 bp for the reference sequence (NM_152274.2). Expression of the gene (by EST data) was found in 27 of 48 adult tissues including kidney, colon,

3rd cervix and uterus, but not heart (NCBI expression viewer, UniGene o Hs.496943). Expression was also noted in 24 of 26 listed tumor tissues http://www.nature.com/naturegenetics small bladder as well as in embryo and fetus. Genes homologous to FAM58A (NCBI HomoloGene: 13362) are found on the X chromosome in the enotes a trait present in the respective case or typicallychimpanzee observed in the overlapping syndromes. and the dog. The zebrafish has a similar gene on d; dupl., duplicated; ESDR, end-stage renal disease; hpl., hypoplastic; hydronephr., hydronephrosis; chromosome 23. However, in the mouse and rat, there are no true homologs. These species have similar but intronless genes on chromo- ASD Rare Occasional Occasional

VU reflux Hydronephr., somes 11 (mouse) and 10 (rat), most likely arising from a retro- transposon insertion event. On the murine X chromosome, the flanking genes Atp2b3 and Dusp9 are conserved, but only remnants of the FAM58A sequence can be detected. FAM58A contains a cyclin-box-fold domain, a protein-binding domain found in cyclins with a role in cell cycle and transcription 3rd 5th X control. No human phenotype resulting from a cyclin gene mutation o Nature Publishing Group Group 200 8 Nature Publishing has yet been reported. Homozygous knockout mice for Ccnd1 Sol. kidney Cros. fused kidneys Pelv. kidney, ESRD Sol. kidney, ESRD Rare Occasional X . © (encoding cyclin D1) are viable but small and have reduced lifespan. valv. pulm. stenosis Bicusp. aortic valve, They also have dystrophic changes of the retina, likely as a result of decreased cell proliferation and degeneration of photoreceptor cells during embryogenesis7,8. Cyclin D1 colocalizes with SALL4 in the nucleus, and both

Supplementary Note cooperatively mediate transcriptional repression9. As the phenotype of 3rd

o our cases overlaps considerably with that of Townes-Brocks syndrome 1 megaureter caused by SALL1 mutations , we carried out co-immunoprecipitation artery stenosis to find out if SALL1 or SALL4 would interact with FAM58A in a manner similar to that observed for SALL4 and cyclin D1. We found that FAM58A interacts with SALL1 but not with SALL4 (Supplemen- tary Fig. 5 online), supporting the hypothesis that FAM58A and SALL1 participate in the same developmental pathway. How do FAM58A mutations lead to STAR syndrome? Growth retardation (all cases; Table 1) and retinal abnormalities (three cases) are reminiscent of the reduced body size and retinal anomalies in cyclin D1 knockout mice7,8. Therefore, a proliferation defect might be partly responsible for STAR syndrome. To address this question, we carried out a knockdown of FAM58A mRNA followed by a proliferation assay. Transfection of HEK293 cells with three different FAM58A-specific RNAi oligonucleotides resulted in a significant reduction of both FAM58A mRNA expression and proliferation of transfected cells (Supplementary Methods and Supplementary Fig. 6 online), supporting the link between FAM58A Radial ray anomaly Congenital heart diseaseHeight (percentile) 3rd PFO, peripheral pulm. TelecanthusLop earsClinodactyly 5th fingerSyndactyly of toes (not 2–3)Anal atresiaGenital anomaly (external)Genital anomaly (internal) XRenal X anomaly Dupl.Urinary vagina tract + anomaly X uterus Hpl. labia X X VU reflux Hpl. labia X X X Hydronephr., VU reflux, X Dupl. vagina, bic. uterus Hpl. labia X X X X Hpl. labia X X Clitoromeg. None X X Clitoromeg. X X None X X X No No X X X Rare No No (low set) Rare X X X Rare Reported in (X) Occasional X lambd., lambdoid; PFO, persistentMore formane detailed ovale; information, pelv., especially pelvic; on pulm., cases pulmonary; 5 sol., and solitary; valv., 6, valvular; is VU, contained vesicourethral. in An the ‘‘X’’ d Table 1 Clinical features in STAR syndrome cases Feature Case1 Case2 Case3Abbreviations: asym., asymmetrical; bic., bicornuate; bicusp., bicuspid; bif., bifid; clitoromeg., clitoromegaly; cor., coronal; cros., crosse Case4 Case5 Case6 EyesCraniosynostosisandcell Cor., lambd. proliferation. Macular hypoplasia Dystr. retina, –5D myopia sagittal Normal

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a c e g L i X:152366805–152692731, 325 kb +4 +2 FAM58A +1 0 UCHL51P BCAP31 –1

–2 ATP2B3 DUSP9 TREX2 BGN PNCK SLC6A8 ABCD1 –4 b d f h 152.36 152.42 152.47 152.53 152.58 152.64 152.69 Mb

c 555+1G>A j c 201dupT c 556-1G>A

1234 5 del 4,249 bp del 40,068 bp

Figure 1 Clinical and molecular characterization of STAR syndrome. (a–f) Facial appearances of cases 1–3 (apparent telecanthus, dysplastic ears and thin upper lips; a,c,e), and toe syndactyly 2–5, 3–5 or 4–5 (b,d,f) in these cases illustrate recognizable features of STAR syndrome (specific parental consent has been obtained for publication of these photographs). Anal atresia and hypoplastic labia are not shown. (g,h) X-ray films of the feet of case 2 showing only four rays on the left and delta-shaped 4th and 5th metatarsals on the right (h; compare to clinical picture in d). (i) Array-CGH data. Log2 ratio represents copy number loss of six probes spanning between 37.9 and 50.7 kb, with one probe positioned within FAM58A. The deletion does not remove parts of other functional genes. (j) Schematic structure of FAM58A and position of the mutations. FAM58A has five coding exons (boxes). The cyclin domain (green) is encoded by exons 2–4. The horizontal arrow indicates the deletion extending 5¢ in case 1, which includes exons 1 and 2, whereas the horizontal line below exon 5 indicates the deletion found in case 3, which removes exon 5 and some 3¢ sequence. The pink horizontal bars above the boxes indicate the http://www.nature.com/naturegenetics amplicons used for qPCR and sequencing (one alternative exon 5 amplicon is not indicated because of space constraints). The mutation 201dupT (case 4) results in an immediate stop codon, and the 555+1G4A and 555-1G4A splice mutations in cases 2, 5 and 6 are predicted to be deleterious because they alter the conserved splice donor and acceptor site of intron 4, respectively.

We found that loss-of-function mutations of FAM58A result in a AUTHOR CONTRIBUTIONS rather homogeneous clinical phenotype. The additional anomalies in S.U. contributed to the clinical evalutation of cases, syndrome delineation and subject enrollment in the study. D.B. performed array CGH, mutation case 1 are likely to result from an effect of the 40-kb deletion on analysis and qPCR. W.B. performed mutation analysis on MYCN, SALL1 expression of a neighboring gene, possibly ATP2B3 or DUSP9.How- and SALL4 and FAM58A breakpoint cloning. F.J.K. performed co-immuno- ever, we cannot exclude that the homogeneous phenotype results from precipitation studies. K. Buiting performed X-chromosome inactivation studies. an ascertainment bias and that FAM58A mutations, including S.K. and P.B. performed cell culture studies, siRNA knockdown experiments and proliferation assays, and contributed to the manuscript. J.B., F.B. missense changes, could result in a broader spectrum of malforma- and A.C. cloned expression constructs and performed RT-PCR. K. tions. The genes causing the overlapping phenotypes of STAR syn- Borowski, K.K.-N., G.M., T.S.-M., B.S., D. Bartholdi, R.S., B.Z., and Nature Publishing Group Group 200 8 Nature Publishing drome and Townes-Brocks syndrome seem to act in the same pathway. A.S.-F contributed to subject enrollment and clinical evaluation. S.U., D.B., © Of note, MYCN, a gene mutated in Feingold syndrome, is a direct J.B., F.J.K., K.Bu., S.K., P.B., R.S., G.M. and A.S.-F. also contributed to the regulator of cyclin D2 (refs. 10,11); thus, it is worth exploring whether manuscript. J.K. oversaw all aspects of the research and wrote major parts of the manuscript. the phenotypic similarities between Feingold and STAR syndrome might be explained by direct regulation of FAM58A by MYCN. Published online at http://www.nature.com/naturegenetics FAM58A is located approximately 0.56 Mb centromeric to MECP2 Reprints and permissions information is available online at http://npg.nature.com/ on Xq28. Duplications overlapping both MECP2 and FAM58A have reprintsandpermissions been described and are not associated with a clinical phenotype in females12, but no deletions overlapping both MECP2 and FAM58A have been observed to date13. Although other genes between FAM58A 1. Green, A., Sandford, R. & Davison, B. J. Med. Genet. 33, 594–596 (1996). 2. Kohlhase, J., Wischermann, A., Reichenbach, H., Froster, U. & Engel, W. Nat. Genet. and MECP2 have been implicated in brain development, FAM58A and 18, 81–83 (1998). MECP2 are the only genes in this region known to result in X-linked 3. Kohlhase, J. et al. Hum. Mol. Genet. 11, 2979–2987 (2002). 4. van Bokhoven, H. et al. Nat. Genet. 37, 465–467 (2005). dominant phenotypes; thus, deletion of both genes on the same allele 5. Spitz, R. et al. Genes Chromosom. Cancer 45, 1130–1142 (2006). might be lethal in both males and females. 6. Carrel, L. & Willard, H.F. Nature 434, 400–404 (2005). 7. Ma, C., Papermaster, D. & Cepko, C.L. Proc. Natl. Acad. Sci. USA 95, 9938–9943 Note: Supplementary information is available on the Nature Genetics website. (1998). 8. Sicinski, P. et al. Cell 82, 621–630 (1995). ACKNOWLEDGMENTS 9. Bohm, J., Kaiser, F.J., Borozdin, W., Depping, R. & Kohlhase, J. Biochem. Biophys. Res. Commun. 356, 773–779 (2007). We thank the research subjects and their families for their participation, generosity 10. Bouchard, C. et al. EMBO J. 18, 5321–5333 (1999). and patience. We thank G. Scherer for critical discussion, P. Hermanns and 11. Knoepfler, P.S., Cheng, P.F. & Eisenman, R.N. Genes Dev. 16, 2699–2712 B. Ro¨sler for help with cell cultures, and C. Lich for technical assistance. (2002). J.K. received funding from the Deutsche Forschungsgemeinschaft (grant no. 12. Van Esch, H. et al. Am. J. Hum. Genet. 77, 442–453 (2005). Ko1850/6-1,6-2). 13. Archer, H.L. et al. J. Med. Genet. 43, 451–456 (2006).

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